Inhibitors of glycogen synthase 3 kinase

ABSTRACT

Compounds of formula 1: 
                         
wherein
         R 1  is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0–3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro;   R 2  is hydroxy, amino, or lower alkoxy;   R 3  is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amino-acyl;   R 4  is H or lower alkyl;
 
and pharmaceutically acceptable salts and esters thereof;
 
are effective inhibitors of GSK3.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of prior application Ser. No.08/948,887, now U.S. Pat. No. 6,153,618 filed Oct. 10, 1997, which inturn claims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication Ser. No. 60/028,306, which was filed Oct. 11, 1996, andwhich is hereby incorporated by reference.

GOVERNMENT LICENSE RIGHTS

The U.S. Government has rights in this invention as provided for by theterms of contract No. DE-AC03-76SF00098 awarded by the Department ofEnergy (DOE) and by the terms of Postdoctoral Fellowships awarded by theNational Science Foundation and the American Cancer Society.

DESCRIPTION

1. Field of the Invention

This invention relates generally to the field of medicinal chemistry,and specifically to compounds which inhibit the activity of glycogensynthase kinase 3 (GSK3).

2. Background of the Invention

Glycogen synthase kinase 3 (GSK3) is a proline-directed serine/threoninekinase originally identified as an activity that phosphorylates glycogensynthase, as described in Woodgett, Trends Biochem Sci (1991) 16:177–81.GSK3 consists of two isoforms, α and β, and is constitutively active inresting cells, inhibiting glycogen synthase by direct phosphorylation.Upon insulin activation, GSK3 is inactivated, thereby allowing theactivation of glycogen synthase and possibly other insulin-dependentevents. Subsequently, it has been shown that GSK3 activity isinactivated by other growth factors or hormones, that, like insulin,signal through receptor tyrosine kinases (RTKs). Examples of suchsignaling molecules include IGF-1 and EGF as described in Saito et al,Biochem J (1994) 303:27–31; Welsh et al, Biochem J (1993) 294:625–29;and Cross et al, Biochem J (1994) 303:21–26. GSK3 has been shown tophosphorylate β-catenin as described in Peifer et al, Develop Biol(1994) 166:543–56.

Other activities of GSK3 in a biological context include GSK3's abilityto phosphorylate tau protein in vitro as described in Mandelkow andMandelkow, Trends in Biochem Sci (1993) 18:480–83; Mulot et al, FEBSLett (1994) 349:359–64; and Lovestone et al, Curr Biol (1995) 4:1077–86;and in tissue culture cells as described in Latimer et al, FEBS Lett(1995) 365:42–46. Phosphorylation of tau and polymerization of thephosphorylated tau is believed to allow formation of paired helicalfilaments that are characteristic of Alzheimer's disease. Thus,inhibition of GSK3 may be useful to treat or inhibit these disorders.

It would be advantageous to develop a method for screening forinhibitors of GSK3 for use and administration in all contexts whereinhibition of GSK3 activity could have a favorable effect.

SUMMARY OF THE INVENTION

We have now invented compounds of formula 1 which inhibit the activityof GSK3:

where R₁ is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, orheteroaralkyl, substituted with 0–3 substituents selected from loweralkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro;R₂ is hydroxy, amino, or lower alkoxy; R₃ is H, lower alkyl, lower acyl,lower alkoxy-acyl, or amino-acyl; R₄ is H or lower alkyl; andpharmaceutically acceptable salts and esters thereof.

Another aspect of the invention is a pharmaceutical composition,comprising a compound of formula 1 and a pharmaceutically acceptableexcipient.

Another aspect of the invention is a method for inhibiting GSK3, bycontacting it with a compound of formula 1.

Another aspect of the invention is a method for treating an indicationmodulated by GSK3, comprising administering a compound of formula 1 to asubject in need thereof.

DETAILED DESCRIPTION

Definitions

The term “compound of formula 1” refers to compounds having the generalstructure:

wherein

-   -   R₁ is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or        heteroaralkyl, substituted with 0–3 substituents selected from        lower alkyl, halo, hydroxy, lower alkoxy, amino, lower        alkyl-amino, and nitro;    -   R₂ is hydroxy, amino, or lower alkoxy;    -   R₃ is H, lower alkyl, lower acyl, lower alkoxy-acyl, or        amino-acyl;    -   R₄ is H or lower alkyl;        and pharmaceutically acceptable salts and esters thereof.

The term “alkyl” as used herein refers to saturated hydrocarbon radicalscontaining from 1 to 12 carbon atoms, inclusive. Alkyl radicals may bestraight or branched. Exemplary alkyl radicals include n-pentyl,n-hexyl, n-octyl, n-dodecyl, 2-dodecyl, and the like. The term “loweralkyl” as used herein refers to straight or branched chain hydrocarbonradicals having from 1 to 8 carbon atoms, such as methyl, ethyl, propyl,isopropyl, n-butyl, s-butyl, t-butyl, n-pentyl, n-hexyl, and the like.“Alkoxy” refers to radicals of the formula —OR, where R is alkyl asdefined above: “lower alkoxy” refers to alkoxy radicals wherein R islower alkyl. “Hydroxy-lower alkyl” refers to radicals of the formulaHO—R—, where R is lower alkylene of 1 to 8 carbons, and may be straightor branched. “Hydroxy-lower alkoxy” refers to radicals of the formulaHO—R—O—, where R is lower alkylene of 1 to 8 carbons, and may bestraight or branched. “Lower alkoxy-lower alkyl” refers to groups of theformula R_(a)O—R_(b)—, where R_(a) and R_(b) are each independentlylower alkyl.

“Alkenyl” refers to hydrocarbon radicals of 2–20 carbon atoms having oneor more double bonds. Alkenyl radicals may be straight, branched, orcyclic. Exemplary alkenyl radicals include 1-pentenyl, 3-hexenyl,1,4-octadienyl, 3,5-diethylcyclohexenyl, and the like. “Lower alkenyl”refers to alkenyl radicals having 2–8 carbon atoms.

The term “alkynyl” refers to hydrocarbon radicals of 2–20 carbon atomshaving one or more triple bonds. Alkynyl radicals may be straight,branched, or cyclic. Exemplary alkynyl radicals include 1-pentynyl,3-hexynyl, octa-2-yn-6-enyl, 3,5-diethylcyclohexynyl, and the like.“Lower alkynyl” refers to alkynyl radicals having 2–8 carbon atoms.

The term “cycloalkyl” refers to alkyl radicals of 3–20 carbon atomshaving at least one ring of carbon atoms, such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, 2-methylcyclopentryl, and the like.“Bicycloalkyl” refers to alkyl radicals of 7–20 carbon atoms having atleast two fused rings of carbon atoms (in which one or more carbon atomsare members of both rings). “Tricycloalkyl” refers to alkyl radicals of7–20 carbon atoms having at least three fused rings of carbon atoms (inwhich one or more carbon atoms of each ring are simultaneously membersof another ring).

The term “haloalkyl” refers to an alkyl radical substituted with one ormore halogen atoms. Exemplary haloalkyl radicals includetrifluoromethyl, 2,2,2-trifluoroethyl, 3-chlorocyclohexyl,2-bromo-3-chlorocyclohexyl, 2,3-dibromobutyl, and the like.

The term “haloalkenyl” refers to an alkenyl radical substituted with oneor more halogen atoms. Exemplary haloalkenyl radicals include3-chloroprop-2-enyl, 4,4-dichlorobut-2-enyl,5-bromo-3-methylcyclohex-2-enyl, and the like.

“Aryl” refers to aromatic hydrocarbons having up to 14 carbon atoms,preferably phenyl or naphthyl. “Aryl-lower alkyl” refers to radicals ofthe form Ar—R—, where Ar is aryl and R is lower alkyl. “Aryloxy” refersto radicals of the form Ar—O—, where Ar is aryl. “Aryloxy-lower alkyl”refers to radicals of the form ArO—R—, where Ar is aryl and R is loweralkyl.

The term “acyl” refers to a radical of the formula RCO—, in which R isH, alkyl as defined above, phenyl, benzyl or naphthyl. Exemplary acylgroups include acetyl, propionyl, formyl, t-butoxycarbonyl, benzoyl, andthe like. “Lower acyl” refers to radicals wherein R is lower alkyl.

The term “halo” refers to a halogen radical, such as F, Cl, Br, or I.

The term “treatment” as used herein refers to reducing or alleviatingsymptoms in a subject, preventing symptoms from worsening orprogressing, inhibition or elimination of the causative agent, orprevention of the infection or disorder in a subject who is freetherefrom. Thus, for example, treatment of non-insulin dependentdiabetes melitis (NIDDM) in a patient may be reduction in the serumlevels of glucose. Treatment of Alzheimer's disease may be halting orretarding the progression of the disease (e.g., as measured by areduction in the rate of dementia).

The term “glycogen synthase kinase 3” or “GSK3” as used herein refers toa protein originally identified by its phosphorylation of glycogensynthase as described in Woodgett et al, Trends Biochem Sci (1991)16:177–81.

The term “biological condition mediated by GSK3 activity” as used hereinrefers to any biological or medical condition or disorder in whicheffective GSK3 activity is identified, whether at normal or abnormallevels. The condition or disorder may be caused by the GSK3 activity ormay simply be characterized by GSK3 activity. That the condition ismediated by GSK3 activity means that some aspect of the condition can betraced to the GSK3 activity. It is expected that by the method of theinvention, inhibiting the GSK3 activity will then prevent, ameliorate ortreat the condition so characterized.

The term “CREB peptide” as used herein refers to a sequence within theCREB DNA-binding protein as described in Wang et al, Anal. Biochem(1994) 220:397–402.

General Methods and Detailed Description

The present invention provides for the inhibition of GSK3 activity,which includes, for example, inhibition of its kinase activity. Byinhibiting GSK3 kinase activity, other activities downstream of GSK3kinase activity are inhibited. For example, inhibition of the GSK3kinase activity can result in the activation of glycogen synthase,because normally GSK3 acts constitutively in cells to inactivateglycogen synthase by direct phosphorylation. When GSK3 kinase activityis inhibited, glycogen synthase may activate leading to a cascade ofevents. GSK3 is also known to act as a kinase in a variety of othercontexts, including but not limited to, for example, phosphorylation ofc-jun, β-catenin, and tau protein. It is understood that inhibition ofGSK3 kinase activity can lead to a variety of effects in a variety ofbiological contexts. The invention is not limited, however, by anytheories of mechanism of how the invention works.

Compounds of the invention may be prepared following standardtechniques. See for example, T. C. Norman et al., J. Am. Chem. Soc.(1996) 118:7430–31, describing the synthesis of olomoucine derivatives.Briefly, compounds may be prepared from 2-amino-6-chloropurine,available, for example, from Aldrich Chemical Co. (St. Louis, Mo.).

Substitutions at R₄ (the N³ position) may be introduced by reacting thestarting material with a strong base (for example NaH) and an alkylhalide (for example, CH₃I) in a solvent such as DMF. The C² amino groupis alkylated by S_(N)2 displacement with a protected 2-haloacetic ester,for example, t-butyl α-iodoacetate, using a strong base (e.g., NaH) inan aprotic solvent such as DMF. The protecting group (e.g., t-butyl) maybe converted to the R₂ substitution, or may be removed to allowimmobilization of the compound on a solid phase for furthermodification. For example, one may treat the ester with trifluoroaceticacid (TFA) and 1,4-dimethoxybenzene, followed by 1 eq PyBroP, 1 eq ofp-nitrophenol, and 3 eq DIEA in CH₂Cl₂, followed by reaction with Rinkresin, 0.06 M DEA, and CH₂Cl₂ to provide the purine derivative coupledto a solid support.

The R₃ substitution may be introduced directly by alkylation (as above)or acylation with a reactive acyl halide (e.g., propionyl chloride). R₃groups bearing reactive moieties may require protection, for example aterminal OH may require protection with a t-butyl group. The C⁶ positionamino group and R¹ substitution may then be introduced by contacting theintermediate with a reagent of the formula R₁—NH₂ (for example,4-trifluoromethylbenzylamine) in DMF/DMSO. The resulting compound maythen be cleaved from the resin, e.g., by treatment with CH₂Cl₂/TFA/Me₂Sat room temperature. The resulting compound (with R₂═NH₂) may bemodified to provide other R₂ substitutions by standard techniques(esterification, etc.).

In presently preferred compounds of the invention, R₁ is either alipophilic alkyl (such as 3-methylbutyl, pentyl, hexyl, cyclohexyl, andthe like, preferably 3-methylbutyl) or a substituted benzyl orpyridylmethyl radical, especially 4-fluorobenzyl, 3-pyridylmethyl,4-pyridylmethyl, 4-trifluoromethylbenzyl, 4-methoxybenzyl, or4-chlorobenzyl. R₂ is preferably NH₂, and R₄ is preferably lower alkyl,particularly methyl. R₃ is preferably H or acyl, optionally substitutedwith NH₂ or CH₃O, especially propionyl, 2-aminoacetyl, 2-methoxyacetyl,3-methylbutyryl, 3-methoxypropionyl, butyryl, or 3-aminopropionyl.

Compounds of the invention are assayed for activity by standardtechniques, preferably employing the GSK3 assay described in theexamples below. The methods include methods to assay for GSK3 kinaseactivity in an in vitro or cell-based assay, a method to assay forinhibitors of binding to GSK3, and an in vivo Drosophila eye screeningassay.

General aspects of the kinase activity assays are conducted as describedin U.S. Pat. No. 4,568,649; EP 154,734; and JP 84/52452, incorporatedherein by reference, which describe kinase activity assays conducted forkinases other than GSK3. It is believed that GSK3 isoforms α and βphosphorylate serine and threonine residues in the amino acid contextserine-proline (SP) or threonine-proline (TP), as well as at theN-terminal serine in the motif SXXXS, provided that the C-terminalserine in this sequence is prephosphorylated, as described in Wang etal, Anal Biochem (1994) 220:397–402 and Roach, J Biol Chem (1991)266:14139–42.

Two of the methods for identifying specific inhibitors of GSK3 are an invitro kinase assay and a cell-based kinase assay. Both kinase assays areperformed similarly, with the distinction between them that the in vitroassay screens inhibitors that will act on the polypeptide GSK3, and thecell-based assay screens in addition for inhibitors that can act withinthe cell at any step in the process of expression of GSK3. Thus, thecell-based assay can screen for, for example, those inhibitors that actduring transcription of GSK3 or that can act during intracellularpost-transcriptional events in the process of making mature GSK3.

For the in vitro form of the assay, recombinant GSK3 is combined withthe other components of the assay including a peptide substrate, and acompound of the invention. The assay published in Wang et al is a GSK3assay makes use of a substrate peptide whose sequence is based on thatof the GSK3 phosphorylation site in the CREB DNA-binding protein. In thepublished assay, the C-terminal serine in the SXXXS motif isprephosphorylated by casein kinase II. However, a modified peptide ofmotif SXXXS having an N-terminal anchor ligand can be synthesized withthe C-terminal serine prephosphorylated (Chiron Mimotopes, Clayton,Australia). The substrate is then able to accomplish both binding to asubstrate anchor at the N-terminal anchor ligand and to eliminate theneed to phosphorylate the C-terminal serine as a separate step. Theanchor ligand is a molecule or mechanism for keeping the substratepeptide present during a wash. For example, where the substrate anchoris biotin, especially in the case where the substrate is bound at theN-terminus to biotin, the anchor can be a molecule that binds biotin,for example, streptavidin.

To conduct the assay using microwells, scintillant may be present bycoating the wells with a phosphorescent material, or by adding it laterin a wash step. The scintillant can be purchased from Packard, Meriden,Conn. Wells coated with scintillant are then coated with streptavidin.Where the scintillant is added later in a wash step, the streptavidincan be present on agarose beads. In any event, the streptavidin in thewells binds the biotin that contacts it. Where the substrate anchor isbiotin, the radiolabel on the phosphorylated substrate that has beenconjugated to the biotin will cause the phosphorescent material tophosphoresce.

Where the streptavidin is attached to phosphorescent agarose beads,binding a biotin-conjugated radiolabeled peptide substrate will causethe beads to phosphoresce and will be an indication of the inhibitoryactivity of the candidate inhibitor. In both the case of the wells linedwith the phosphorescent material, and the agarose beads, a reduction inphosphorescence as compared to a control amount of phosphorescencemeasured under non-inhibitory conditions, indicates the presence of afunctional inhibitor of GSK3 activity. If the peptide has beenphosphorylated by GSK3 with ³²P-labeled or ³³P-labeled phosphate,radioactive decay will cause the phosphorescent material present in amicrowell or mixed in agarose beads that are present in the reactionmixture to emit light and the measure of the amount of light emittedwill be a measure of the activity of GSK3 in the assay. Low activity ofGSK3 observed in the presence of a candidate inhibitor, as compared tothe activity of GSK3 in the absence of the inhibitor, may indicate thatthe inhibitor is functional and can inhibit GSK3 kinase activity. In anycase, an equal amount of streptavidin should be loaded into each well orshould be affixed to the agarose beads, and an equal amount of the beadsadded to each assay.

The cell-based assay includes in addition, a cell that can express GSK3,such as for example a cell transformed with the gene encoding GSK3,including regulatory control sequences for the expression of the gene.For conducting the cell-based assay, the cell capable of expressing GSK3is incubated in the presence of a compound of the invention, the cell islysed and the GSK3 is immunoprecipitated or otherwise purified, and thepurified GSK3 is placed in contact with a peptide substrate, andradiolabeled phosphate-ATP. The amount of phosphorylation of thesubstrate is an indication of the degree of inhibition accomplished bythe compound of the invention. During the cell-based assay, inhibitionof GSK3 activity may occur either by inhibiting the expression of GSK3or by inhibition of GSK3's protein kinase activity, both of which willbe indicated by phosphorylation (or lack thereof) of the substratepeptide in the cell-based assay. However, one can determine which aspectis inhibited by assaying inhibition of GSK3 in vitro as described above.

An alternative assay that can be used to screen in vivo for inhibitorsof GSK3 kinase activity is a Drosophila eye screen for inhibitors. Thefly eye screen detects inhibitory activity by expressing GSK3 inDrosophila under the control of an eye-specific promoter. The eyespecific promoter can be any promoter specific to expression of proteinsin eye tissue, including but not limited to, for example, GMR asdescribed in Hay et al, Development (1994) 120:2121–29, and thesevenless promoter, as described in Bowtell et al, Genes and Development(1988) 2:620–34. The screening assay for inhibitors of GSK3 activity isthen conducted by feeding the flies food containing a compound of theinvention. If the inhibitor is functional, the eye morphology revertsfrom mutant to wildtype. The expression of GSK3 under the control of theeye specific promoter leads to developmental defects which result inobvious aberrations in the external morphology of the external eyetissue. The mutant morphology that results in these transgenic flies iscalled “roughening”. These defects may depend on GSK3 activity, asindicated by a control experiment using developing flies expressing aGSK3 mutant that contains a mutated kinase domain. The fly eye cellstransformed with a catalytically inactive GSK3 mutant are incapable ofeliciting the rough eye morphological effects of the catalyticallyactive counterpart.

Drosophila embryos are transformed by the method of Karess and Rubin,Cell (1984) 38:135–46 with a polynucleotide construct comprising a GSK3coding sequence under the regulatory control of a GMR promoter. Theflies are allowed to develop normally and are selected by eye morphologyfor successful transformants. Successful transformants have an aberrantmorphology characterized by rough eye cell morphology that is detectableunder a dissecting microscope. The transgenic flies are then fed foodspiked with an appropriate dose of a compound of the invention. Theamount of the inhibitor will depend on the deduced possible potency ofthe molecule as an inhibitor. The flies are fed a compound of theinvention throughout third instar larval development, during which timethey are observed for reversions of their eye morphology to wildtype ornormal. Positives are identified. This method may also be applied as aprimary screen to identify additional compounds of the invention thathave positive activity. Variations to the protocol include injecting acompound of the invention into the third instar larvae of thetransformants which are then observed for a reversion of the rough eyemorphology to normal.

The compounds of the invention may be administered by a variety ofmethods, such as intravenously, orally, intramuscularly,intraperitoneally, bronchially, intranasally, and so forth. Thepreferred route of administration will depend upon the nature of thecompound and the condition to be treated. Compounds may be administeredorally if well absorbed and not substantially degraded upon ingestion(compounds of the invention are generally resistant to proteases). Thecompounds may be administered as pharmaceutical compositions incombination with a pharmaceutically acceptable excipient. Suchcompositions may be aqueous solutions, emulsions, creams, ointments,suspensions, gels, liposomal suspensions, and the like. Thus, suitableexcipients include water, saline, Ringer's solution, dextrose solution,and solutions of ethanol, glucose, sucrose, dextran, mannose, mannitol,sorbitol, polyethylene glycol (PEG), phosphate, acetate, gelatin,collagen, Carbopol®, vegetable oils, and the like. One may additionallyinclude suitable preservatives, stabilizers, antioxidants,antimicrobials, and buffering agents, for example, BHA, BHT, citricacid, ascorbic acid, tetracycline, and the like. Cream or ointment basesuseful in formulation include lanolin, Silvadene® (Marion), Aquaphor®(Duke Laboratories), and the like. Other topical formulations includeaerosols, bandages, sustained-release patches, and the like.Alternatively, one may incorporate or encapsulate the compound in asuitable polymer matrix or membrane, thus providing a sustained-releasedelivery device suitable for implantation near the site to be treatedlocally. Other devices include indwelling catheters and devices such asthe Alzet® minipump. Further, one may provide the compound in solidform, especially as a lyophilized powder. Lyophilized formulationstypically contain stabilizing and bulking agents, for example humanserum albumin, sucrose, mannitol, and the like. A thorough discussion ofpharmaceutically acceptable excipients is available in Remington'sPharmaceutical Sciences (Mack Pub. Co.).

EXAMPLES

The examples presented below are provided as a further guide to thepractitioner of ordinary skill in the art, and are not to be construedas limiting the invention in any way.

Example 1 Preparation of Compounds

Compounds of the invention were prepared following the method disclosedin T. C. Norman et al., J. Am. Chem. Soc. (1996) 118:7430–31.

(A) 2-Amino-6-chloropurine was treated with NaH (1.1 eq) and CH₃I (1 eq)in DMF. The product was treated with trifluoroacetic anhydride (3 eq) inCH₂Cl₂, then alkylated with t-butyl α-iodoacetate (2 eq) and NaH (1.1eq) in DMF. The reaction was quenched with K₂CO₃ in MeOH. The productwas then treated with trifluoroacetic acid (TFA) and1,4-dimethoxybenzene, followed by PyBroP (1 eq), p-nitrophenol (1 eq),and DIEA (3 eq) in CH₂Cl₂, and coupled to Rink-derivatized polyethylenecrowns (Chiron Mimotopes, Clayton, Australia), 0.06 M DIEA, and CH₂Cl₂to provide the purine derivative coupled to a solid support (R₄=methyl).

The intermediate was acylated with 2-methoxyacetyl chloride (0.2 M) inthe presence of 4-methyl-2,6-di-t-butylpyridine (0.25 M) in CH₂Cl₂ at37° C. for 12 hours (R₂=CH₃OCH₂CO). The resulting intermediate wasreacted with 4-trifluoromethyl-benzylamine (0.25 M) in DMF/DMSO (1:1 byvolume) at 4° C. for 16 h, followed by cleavage from the support bytreatment with CH₂Cl₂/TFA/Me₂S at room temperature for 2 hours, toprovide CHIR 21208:

(B) Proceeding as in part (A) above, the following compounds were made:

Compound ID R₁ R₂ R₃ R₄ 21172 F₃C-φ-CH₂—(*) NH₂ H CH₃ 21232 F₃C-φ-CH₂—NH₂ NH₂CH₂CO— CH₃ 21220 F₃C-φ-CH₂— NH₂ CH₃CH₂CO— CH₃ 209572-pyridylmethyl NH₂ H CH₃ 20981 2-pyridylmethyl NH₂ (CH₃)₂CHCH₂CO CH₃21005 2-pyridylmethyl NH₂ CH₃CH₂CO— CH₃ 21132 (CH₃)₂CHCH₂CH₂ NH₂CH₃OCH₂CO CH₃ 21131 4-F-φ-CH₂— NH₂ CH₃OCH₂CO CH₃ 21196 F₃C-φ-CH₂— NH₂(CH₃)₂CHCH₂CO CH₃ 21095 4-F-φ-CH₂— NH₂ H CH₃ 21100 4-CH₃O-φ-CH₂ NH₂ HCH₃ 20951 cyclohexyl NH₂ H CH₃ 20956 4-pyridylmethyl NH₂ H CH₃ 210044-pyridylmethyl NH₂ CH₃(CH₂)₂CO CH₃ 21075 4-Cl-φ-CH₂ NH₂ CH₃(CH₂)₂CO CH₃21156 (CH₃)₂CH(CH₂)₂ NH₂ NH₂(CH₂)₂CO CH₃ (*)φ = phenyl

Example 2 Assay

(A) A GSK3β gene was created in which a haemagluttinin (HA) epitope wasfused to the N-terminal end of the GSK3β open reading frame in plasmidvector pCG, a pEVRF derivative, described in Giese et al. Genes&Development (1995) 9:995–1008, and in Matthias et al., Nucleic AcidsRes. (1989) 17:6418. pCG has a modified polylinker, and directsexpression in mammalian cells from the human cytomegaloviruspromoter/enhancer region. The resulting plasmid is pCG-HA-GSK3β.pCG-HA-GSK3β was transiently transfected into COS cells on 10 cm tissueculture plates using DEAE-Dextran, as described in Ausubel et al (1994)“Current Protocols In Molecular Biology”, (Greene Publishing Associatesand John Wiley & Sons, New York, N.Y.).

The final density of cells was 70% confluent and these cells were lysedin 700 μl Triton lysis buffer (20 mM Tris HCl, pH 7.9, 137 mM NaCl, 1.0%Triton® X-100, 10% glycerol, 1 mM NaVO₃, 20 mM NaF, 30 mM pNpp, 15 mMPPi). Anti-HA antibody (12CA5 monoclonal antibody purchased fromBoehringer Mannheim, Indianapolis, Ind.) was added to 300 μl of thislysate to a final concentration of 4 μg/ml and incubated for 1 h at 4°C. 100 μl of a 50% slurry of protein A-Sepharose® beads were added for 2h at 4° C.

The beads were pelleted by centrifugation for 10 seconds in amicrocentrifuge and washed with 0.5 M LiCl, 0.5% Triton® X-100, twicewith phosphate buffered saline (PBS) and once with 10 mM Tris HCl, pH7.5, 5 mM MgCl₂, 1 mM DTT. All wash buffers contained 1 mM NaVO₃ and 25mM β-glycerolphosphate. 33 μl of the beads were analyzed by SDS-PAGE andwestern blotting with anti-HA antibody (12CA5) to quantitate the amountof HA-GSK3βpresent.

The remaining 17 μl of beads was assayed according to the protocol ofWang et al, Anal Biochem (1994) 220:397–402. To each 17 μl of pellet wasadded 3 μl 10× GSK buffer (100 mM MgCl₂, 20 mM DTT, 3M Tris HCl, pH7.5), 0.7 μl CREB peptide (either prephosphorylated ornon-prephosphorylated, 5 mg/ml, Chiron Mimotopes Peptide Systems, SanDiego, Calif.), 0.3 μl 10 mM rATP, 1 μl γ³²P-ATP (6000 Ci/mmol), 0.06 μl5 mg/ml of protein kinase inhibitor (a protein kinase-A inhibitor orPKI), and 25 μl H₂O. The reaction was allowed to proceed for 20 min at22° C. and then was stopped with 8 μl 500 mM EDTA. 22 μl of eachreaction was spotted onto P81 phosphocellulose filter paper (purchasedfrom Gibco-BRL Life Technologies, Gaithersburg, Md.) washed 4 times for5 minutes in 75 mM H₃PO₄ The filter papers were then assayed in ascintillation counter. Filter papers from experiments using theprephosphorylated CREB peptide substrate yielded counts of 85,000±5,000cpm/min, whereas filter papers from experiments using thenon-prephosphorylated CREB peptide substrate yielded counts of5,000±1,000 cpm/min. The results indicated that the substrate wasphosphorylated by GSK3 in the absence of an inhibitor, although thecontrol substrate was not phosphorylated. This experiment demonstratesthe specificity of the peptide as a GSK3 substrate.

(B) The compounds prepared in Example 1 above were assayed for activityas follows:

A 96-well round bottom plate was blocked by incubation with 400 μl/well1% BSA/PBS for 60 min at room temperature. The blocking reagent was thenaspirated. An enzyme/substrate buffer was prepared (1.225 ml 1MTris-HCl, pH 7.5, 0.41 ml 1M MgCl₂, 41 μl DTT, 250 μl 500 μg/ml GSK-3β,9.5 μl 5 mg/ml biotin-phosphopeptide, 33.1 ml 1% BSA/PBS), and 300 μlenzyme/substrate/buffer was added to each well.

Varying concentrations of each compound were added to individual wells,or staurosporine in DMSO (final concentrations of staurosporine 100 nMor 20 nM). Next 50 μl/well ATP mixture (3.85 μl 10 mM cold ATP, 8 μl hotATP, 5.5 ml H₂O) was added, and the reaction allowed to proceed for 180min at room temperature.

Three streptavidin-coated Labsystems Combiplate 8 plates were blockedwith 1% BSA/PBS, 300 μl/well, for ≧60 min at room temperature, and theblocking reagent aspirated. Stopping reagent (50 μM ATP, 20 mM EDTA)(100 μl/well) was added to the streptavidin-coated plates, and 100 μl ofthe enzyme reaction mixture transferred to the streptavidin-coatedplates in triplicate. The plates were incubated at room temperature for60 min, and washed 5× with PBS using a Corning plate washer. Finally,Microscint-20 scintillation fluid (200 μl/well) was added to the plates,the plates sealed, and after incubating for 30 min, counted on aTopCount counter. The results were as follows:

Compound % inhibition at 1 μM 21208 63 21172 61 21232 58 21220 53 2095750 20981 47 21005 45 21132 45 21131 43 21196 43 21095 41 21100 41 2095140 20956 40 21004 40 21075 40 21156 40

The results demonstrated that the compounds of the invention wereeffective in inhibiting the kinase activity of GSK3.

1. A compound of formula 1:

wherein: R₁ is aryl, substituted with 0–3 substituents selected fromlower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, andnitro; R₂ is hydroxy, amino, or lower alkoxy; R₃ is H, lower acyl, loweralkoxy-acyl, or amino-acyl; R₄ is H or lower alkyl; and pharmaceuticallyacceptable salts thereof.
 2. The compound of claim 1 wherein R₄ ismethyl.
 3. The compound of claim 2, wherein R₂ is amino.
 4. Apharmaceutical composition, comprising: a compound of the formula 1:

wherein R₁ is aryl, substituted with 0–3 substituents selected fromlower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, andnitro; R₂ is hydroxy, amino, or lower alkoxy; R₃ is H, lower acyl, loweralkoxy-acyl, or amino-acyl; R₄ is H or lower alkyl; or apharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable excipient.
 5. The compound of claim 3, wherein R₃ ispropanoyl.
 6. The compound of claim 3, wherein R₃ is 3-methylbutanoyl.7. The compound of claim 3, wherein R₃ is butanoyl.
 8. The compound ofclaim 3, wherein R₃ is H.
 9. The compound of claim 3, wherein R₃ isaminoacetyl.
 10. The compound of claim 3, wherein R₃ is methoxyacetyl.